Ehrlichia canis genes and vaccines

ABSTRACT

This invention provides the sequence of 5,299 nucleotides from the  E. canis  genome. There are four proteins, ProA, ProB, MmpA, and a cytochrome oxidase homolog, as well as a partial lipoprotein signal peptidase homolog at the carboxy terminus, coded for in this cloned fragment. The antigenic properties of these proteins allow them to be used to create a vaccine. An embodiment of this invention includes the creation of a DNA vaccine, a recombinant vaccine, and a T cell epitope vaccine. Another embodiment of this invention includes the use of serological diagnosis techniques.

FIELD OF THE INVENTION

The invention pertains to the field of veterinary pathogens. Moreparticularly, the present invention pertains to the sequence of specificgenes of the bacterial canine pathogen Ehrlichia canis and theapplication of this technology to the development of a vaccine.

BACKGROUND OF THE INVENTION

The present invention relates to the sequence of genes from the E. canisbacterium, and the development of a vaccine against this organism.

Ehrlichia canis (E. canis) is a small gram-negative, obligatelyintracytoplasmic rickettsia. This bacteria is the agent which causescanine monocytic ehrlichiosis (CME), a tick-borne disease whichpredominantly affects dogs. The most common carrier of E. canis is thebrown dog tick Rhipicephalus sanguineus. The disease was describedoriginally in Algeria in 1935. It was subsequently recognized in theUnited States in 1962, but is now known throughout much of the world.Canine monocytic ehrlichiosis caused much concern during the VietnamWar, when 160 military dogs died from the E. canis infection. There isno vaccination currently available against E. canis. It is a lifethreatening disease that continues to be an important health concern forveterinarians and pet owners alike.

Canine monocytic ehrlichiosis is an infectious blood disease. Areduction in cellular blood elements is the primary characteristic ofthe disease. E. canis lives and reproduces in the white blood cells(leukocytes). It eventually affects the entire lymphatic system, anddevastates multiple organs. By targeting the white blood cells, thesecells die off rapidly. These dead blood cells migrate primarily to thespleen, which enlarges as a result. The bone marrow recognizes the lossof the white blood cells and works to form new, healthy cells. It sendsout the cells prematurely, and these immature cells do not workproperly. Often, these immature cells mimic those in leukemic patients,so the disease is misdiagnosed as leukemia. Canine monocyticehrlichiosis may also predispose dogs to various cancers.

There are three stages of canine monocytic ehrlichiosis. The first,acute stage mimics a mild viral infection. During the acute stage, most,if not all, of the damage is reversible and the animal is likely torecover. This is the stage where treatment is the most effective,stressing the need for early detection. Without treatment, however, theanimal will progress into a subclinical (second) stage and/or to thechronic (final) stage. When the animal has reached the chronic stage,the bacterial organism has settled within the bone marrow. Many dogs inthis stage suffer massive internal hemorrhage, or develop lethalcomplications such as sudden stroke, heart attack, renal failure,splenic rupture or liver failure.

E. canis can be cultured in vitro in a mammalian-derived cell line(DH82). Continued maintenance of these cells is difficult because thecell culture must be supplemented with primary monocytes (white bloodcells found in bone marrow) every two weeks. The cultures are very slowgrowing, and the culture media is expensive.

Data concerning the genes in the E. canis genome has concentratedprimarily on the 16S rRNA gene. Previous work has sequenced this gene,which is a ubiquitous component of the members of the ehrlichia family,as well as the majority of organisms worldwide. The high sequencehomology between this gene throughout the living world and its poorimmunogenicity makes it an unsuitable candidate for vaccine development.It is necessary to find other genes within this genome if hope for avaccine against this deadly disease can ever be realized.

Sequencing of the 16S rRNA gene indicates that E. canis is closelyrelated (98.2% homology) to E. chaffeensis, the novel etiologic agent ofhuman ehrlichiosis. Western blots of E. canis are similar when probedwith antisera to E. canis, E. chaffeensis, and E. ewingi (another causeof human ehrlichiosis) indicating a close antigenic relationship betweenthese three species (Chen et al., 1994).

The indirect fluorescent antibody test (IFA) has been developed fordetecting canine monocytic ehrlichiosis. IFA detects the presence ofantibodies against the invading organism in a dog's blood.Unfortunately, this test is not always accurate. Sometimes, dogs willtest negative in the acute phase because their immune system is delayedin forming antibodies. Another false negative may occur if there is alow titer in the chronic stage. An additional drawback of this test isthe cross-reactivity found. The anti E. canis polyclonal antibodypositively reacts with E. chaffeensis, undermining the specificity ofthe test. An alternative test, the Giesma smear, has been used to locatethe actual organism in a dog's blood. Unfortunately, despite appropriatestaining techniques and intensive film examination, the organismsfrequently can not be located. The fallibility of these tests makes itessential to provide better diagnostic tools for this disease.

Due to difficulties in the detection of a tick bite, early diagnosis ofinfection, the suppression of host defenses and the nature of persistentinfection of the disease, an effective vaccine against E. canis isurgently needed for dogs.

SUMMARY OF THE INVENTION

This invention discloses novel sequence data for E. canis genes.Specifically, a clone has been identified and sequenced. Four proteinstermed ProA, ProB, mmpA (for morula membrane protein, which is an ORF),and a cytochrome oxidase homolog, have been identified within thisclone. In addition, a partial gene encoding a lipoprotein signalpeptidase homolog has been discovered.

An embodiment of this invention includes the creation of a vaccine withthis sequence and protein information. The proteins disclosed in thisinvention are extremely antigenic. Therefore, they have the potential tobe extremely useful as a vaccine. The types of vaccine made available bythis novel technology include a DNA vaccine, a recombinant vaccine, anda T cell epitope vaccine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the three clones identified in the library screen.

DESCRIPTION OF THE PREFERRED EMBODIMENT

E. canis causes a devastating canine disease. Currently, there is novaccine available to prevent this disease. This invention provides thetools necessary to develop such a vaccine. More specifically, four geneshave been identified from a genomic fragment of E. canis, named ProA,ProB, mmpA, and a cytochrome oxidase homolog. In addition, a partialgene coding for a lipoprotein signal peptidase homolog has been found.Any of these proteins can be utilized in an embodiment of this inventionto develop a vaccine.

Screening an E. canis Library

To identify genes in the E. canis genome, a genomic DNA expressionlibrary was constructed. An E. canis strain isolated from dogs withcanine ehrlichiosis was grown in the dog cell line DH82 by a techniquebeing known in the art, and incorporated by reference (Dawson et al.,1991; Rikihisa, 1992). The cells were harvested and the chromosomal DNAextracted as described by a technique known in the art (Chang et al.,1987; Chang et al., 1989a; Chang et al., 1989b; Chang et al., 1993a;Chang et al., 1993b). To construct the library, 200 μg of DNA waspartially digested with Sau3A. DNA fragments from 3 to 8 kb wereisolated and ligated to a plasmid, pHG165 (Stewart et al., 1986). Theplasmids were transformed into E. coli TBI (Chang et al., 1987).

The library was screened with polyclonal antibodies against E. canis.Polyclonal antibodies were generated from dogs that had been bitten by atick harboring E. canis. The polyclonal antibodies were preabsorbed withthe lysate of an E. coli host strain. The library was plated on petriplates at a density of 1,000 colony forming units. Colonies weretransferred to nitrocellulose and each filter was probed with 1 ml ofthe preabsorbed polyclonal antibodies. Positive colonies were identifiedwith a second antibody consisting of an alkaline phosphatase-conjugatedgoat anti-rabbit IgG (Kirkegaard and Perry Laboratories, Gaithersburg,Md.), followed by color development with a substrate solution containingnitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate(BCIP). Positive clones were rescreened three times.

Three clones were isolated from this screening procedure, pCH2, 4, and 5(FIG. 1). Within these three positive clones, an overlapping readingframe was found that codes for mmpA. The longest genomic fragment (pCH4)encodes four complete genes and one partial gene. It completely encodesthe proteins ProA, ProB, mmpA, and a cytochrome oxidase homolog, as wellas containing the partial sequence of a lipoprotein signal peptidasehomolog. ProA and ProB are located on a single operon. Restrictionendonuclease digestion mapping and DNA sequencing were done bytechniques known in the art, and incorporated by reference (Chang et.al., 1987; Chang et. al., 1989a; Chang et. al., 1989b; Chang et. al.,1993a; Chang et. al., 1993b). Briefly, the DNA sequence was determinedby automated DNA sequencing on the ABI PRISM Model 377 DNA system. Thecomplete nucleotide sequences were determined on both strands by primerwalking. The thermal cycling of the sequencing reactions utilized theTaq Dye Deoxy™ Terminator Cycle sequencing kit. Databases were searchedfor homologous proteins through the use of the BLAST network service ofthe National Center for Biotechnology Information (NCBI) (Althchul etal., 1990; Gish et al., 1993).

Sequence Information

The E. canis genes were sequenced. The cloned fragment contains 5,299nucleotides, and codes for four proteins. There is also one partial geneat the carboxy terminus. SEQ. ID. NO. 1 is the entire nucleotidesequence. SEQ. ID. NO. 2and 3 are the translation of nucleotides 12through 533 from SEQ. ID. NO. 1 and code for a cytochrome oxidasehomolog, which was deemed ec3 and encodes a product of 174 amino acids.Cytochrome oxidase is important in virulence, and therefore is a strongcandidate for use in a vaccine. SEQ. ID. NO. 4 and 5 are the translationof nucleotides 939 through 2,252 from SEQ. ID. NO. 1 and code for ProA.ProA begins 402 nucleotides downstream from the end of ec3 and encodes aproduct of 438 amino acids. The ProA protein shares extensivecharacteristics with the putrilysin family, which is a subset ofmetallopeptidases. The highest homology is seen in the N-terminalportion, which includes conserved putative metal binding sequence,His-X-X-Glu-His as well as conserved Glu for catalysis. ProA was alsoshown to share 27% of its characteristics over 284 amino acids withMPP-β. (mitochondrial processing peptidase) in Solanum tuberosum(Genbank accession number X80237) and 27% of its characteristics over222 amino acids with E. Coli pitrilysin (Genbank accession numberM17095). Many bacterial zinc proteases are also found to share homologywith ProA protein, such as PqqF in M. extrorquens AMI where 36% of 376amino acids are shared(Genbank accession number L43135), Y4wA inRhizobium sp. where 34% of 416 amino acids are shared (Genbank accessionnumber AE000103), 27% of 419 amino acids of the putative gene productPA0372 in Pseudomonas aeruginosa (Genbank accession number AE004475),and 26% of 389 amino acids of the putative gene product of HP1012 inHelicobacter pylori, strain 26695 are shared (Genbank accession numberAE000609). SEQ. ID. NO. 6 and 7 are the translation of nucleotides 2,258through 3,610 from SEQ. ID. NO. 1 and code for ProB. ProB begins 6nucleotides after the end of proA and encodes a protein of 451 aminoacids long. Through Blast analysis it was shown that ProB does notcontain the conserved Zinc- binding domain. PrOB was shown to sharehomology with both subunits of the MPP subfamily and some bacterialputative zinc proteases such as PqqF, Y4wA, and the gene products ofPA0372. ProB also shared homology with bacterial proteases that do notcontain Zinc-ligand motif, but show similarities with the pitrilysinfamily, such as PqqG, Y4wB, gene product of PA0371, and the gene productof HP0657 in M. extrorquens AMI, Rhizobium sp., P. aeruginosa, and H.pylori 26695. Preliminary evidence indicates that ProA and ProB areproteases. SEQ. ID. NO. 8 and 9 are the translation of nucleotides 4,132through 4,794 from SEQ. ID. NO. 1 and code for ORF, designated mmpA. Thepolypeptide that is generated consists of 221 amino acids and does nothave a significant identity to any proteins found in existing databases.SEQ. ID. NO. 10 and 11 are the translation of the complementary sequenceof nucleotides 4,883 through 5,299 from SEQ. ID. NO. 1 and code for thepartial sequence of a lipoprotein signal peptidase homolog. Lipoproteinsignal peptidases are membrane proteins, and by nature may be lessdesirable for vaccine development. However, this protein is still worthpursuing in the creation of a vaccine.

Structure and Expression of ProA, ProB, mmpA, Cytochrome Oxidase and theLipoprotein Signal

Structurally, MmpA is extremely hydrophobic with five transmembranesegments and three potential antigenic determinants, which are proteinkinase C phosphorylation sites located in position 7 (Ser), 37 (Ser),and 213 (Ser) and one possible casein kinase II phosphorylation site inposition 177 (Thr), where the above determinants were characterized byPROSITE and the PCgene programs. MmpA was localized primarily in themorula membrane. Since MmpA contains three potential phosphorylationsites, this indicates the possibility of a similar communication systemas seen by phosphorylation of one of the chlamydial inclusion membraneproteins, IncA. E. canis grown in vitro (DH82 cells) expressed MmpA,furthermore, sera obtained from dogs that were naturally infected andexperimentally infected with E. canis recognized MmpA, which confirmsthat in vivo and in vitro expression of MmpA as well as the antigenicityof MmpA. The above two results indicate that MmpA is capable ofstimulating an immune response, which is necessary for a vaccine to beeffective. However, E. canis is an intracellular organism, cell mediatedimmunity is more important in protecting the dog against this type ofinfection then humoral immunity and it may be possible to direct theseantigens toward a predominant Th1 response using an appropriateadjuvant. The mmpA gene was found in E. canis and E. chaffeenis but wasnot present in the HGE agent. However, the MmpA protein was notexpressed by E. chaffeenis on a western blot. E. canis with MmpA causedcells to lyse, indicating the presence of MmpA protein, where E.chaffeenis with MmpA did not lyse. This result lends to the conclusionthat the MmpA protein may be useful for serodiagnosis in differentiatingE. canis and E. chaffeenis. Furthermore, MmpA, ProA, and ProB proteinscan be used as antigens in ELISA or Western blot analysis to perform adiagnosis of an E. canis infection in animals.

Structurally, ProA and ProB are very similar except for the fact thatProA contains a catalytic zinc-binding motif and ProB does not containany catalytic residues. ProA and ProB were localized to the solublecytoplasmic and periplasmic protein portion, where a tiny amount of ProAwas detectable in the inner membrane fraction of the bacterial fractionsthat were collected to do subcellular fractionation to determine asubcellular location. E. canis and E. chaffeenis infected DH82 cellsboth lysed that contained anti-rProA antibodies, showing that both E.canis and E. chaffeenis express ProA in culture. Furthermore, bothnaturally and experimentally infected dogs with E. canis infectedDH82-cells recognize rProA and rProB lending to the conclusion that ProAand ProB are expressed in vivo and in vitro. However, ProB was notdelectable in a western blot using anti-rProB antibodies with E.chaffeenis. E. canis did detect anti-rProB antibodies. This result showsthat ProB may serve as a tool for serological differentiation of E.canis and E. chaffeenis. Antisera from naturally and experimentallyinfected dogs with E. canis contained antibodies recognizing rProA andrProB. Serum from an uninfected dog did not recognize either of the twoproteins. Immunofluorescence staining of E. canis in DH82 cells withrabbit anti-rProA arid anti-rProB sera was performed, both ProB and ProAantiserum strongly label the intracellular ehrlichial organisms, showingthat ProA and ProB can serve as target antigens and that anti-rProA andanti-rProB sera can be used for indirect immunofluorcscent assays (IFA)diagnosis. Recombinant ProB can be used as an antigen in ELISA orWestern blot analysis to perform a diagnosis of an E. canis infection inanimals.

Overexpression of ProA, ProB, ORF, Cytochrome Oxidase and theLipoprotein Signal Peptidase Homolog

The E. canis antigens are overexpressed in a T7 promoter plasmid. ThepRSET vector allows high level expression in E. coli in the presence ofT7 RNA polymerase, which has a strong affinity for the T7 promoter.After subcloning the antigen genes into the pRSET vector, the subclonesare transformed into an F′ E. coli JM109 strain. For maximum proteinexpression, the transformants are cultured to O.D. 600=0.3, exposed toIPTG (1 mM) for one hour and then transfected with M13/T7 bacteriophagesat a multiplicity of infection (MOI) of 5-10 plaque forming units (pfu)per cell. Time course studies indicate that maximum induction is reachedtwo hours after induction.

The pellet is harvested by centrifugation and the cells are resuspendedin 6M Guanidinium (pH 7.8). Cells are ruptured by French press and thetotal lysate is spun at 6000 rpm to separate cell debris by a techniqueknown in the art, and hereby incorporated by reference (Chang et al.,1993c). Immobilized metal ion affinity chromatography (IMIAC) is used topurify each of the proteins under denaturing conditions as described bythe manufacturer (Invitrogen, San Diego, Calif.). The protein samplesare separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) andvisualized after staining with coomassie blue.

Diagnosis Techniques: KELA ELISA. Western Blotting (Immunoblots), andPCR

The recombinant ProA, ProB, and MmpA proteins are useful diagnosticagents. One diagnostic technique where the ProA, ProB, and MmpA proteinsare used is kinetic enzyme linked immunosorbent assay (KELA ELISA), asdescribed by a technique known in the an (Appel et al., 1993; Chang etal., 1995). KELA measures the levels of serum antibodies to E. canisthat is present. In this diagnostic technique, diluted serum (1:100dilution) is added to duplicate wells in microliter plates that containantigens of MmpA, ProA, and ProB. The antigens are prepared byFrench-pressing them. The bound antibodies are then detected by usingsecond antibodies of a goat anti-canine antibody of heavy and lightchain specificity conjugated to horseradish peroxidase (HRP). Colordevelopment is seen and measured using the chromogentetramethylbenzidine with H₂O₂ as a substrate, which is measuredkinetically and expressed as the slope of the reaction rate between theenzyme and substrate solution. Each unit of slope is designated as aKELA unit. The cutoff point between positive and negative samples isthen confirmed by Western blotting against French-pressed E. canis.

The procedure for Western blot analysis, as described by a techniqueknown in the art (Appel et al., 1993; Chang et al., 1995), is performed.Recombinant ProA, ProB, and MmpA are used as antigens and are subjectedto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Western blot analysis is performed in a miniblotter. Test sera fromexperimental animals are used as a first antibody, followed by goatanti-dog IgG conjugated to HRP as a second antibody. Bands are developedby using substrates, such as 24 μg of 4-chloro-1-naphthol in 8 ml ofmethyl alcohol, 40 ml of Tris-buffer solution, and 24 μl of 30% H₂O₂.

Another diagnostic technique recombinant ProA, ProB, and MmpA proteinscan be used for is PCR diagnosis. The DNA from biopsy samples of skin orfrom post mortem tissues or blood are extracted as described by atechnique known in the art (Chang et al., 1998a; Chang et al., 1998b).To prevent contamination of the mixtures and samples, DNA extraction,amplification, and detection of PCR products are all performed indifferent rooms. Amplification of E. canis MmpA, ProA, or ProB-specifictarget sequences is carried out in a 50-μl reaction mixture. As apositive control, E. canis genomic DNA is used. As a negative controldistilled water is used. The reaction mixture is then put through 40cycles of amplification using an automated DNA thermal cycler. Eachcycle involves healing the reaction mixture to 94° C. from 1 minute, tocause the DNA to denature; cooling of the reaction mixture to 69° C. for1 minute, to allow the primers to anneal; and then heating the reactionmixture to 72° C. for 2minutes, to allow primer extension to occur. Gelelectrophoresis on a 1.5% agarose gel is done in order to getvisualization of the PCR amplification products.

Vaccine Development

Prior to the present invention, no vaccine against E. canis had beendeveloped. E. canis is endemic in dogs and closely related canidae inmany parts of the world. Dogs in North America are also increasingly atrisk and the application of the present invention can potentially savethe lives of thousands of dogs each year. An E. canis vaccine that canelicit cell-mediated immunity against this tick-borne disease of dogs isdesperately needed.

DNA Vaccine

A DNA vaccine is constructed by subcloning the gene of interest into aeukaryotic plasmid vector. Candidate vectors include, but are notlimited to, pcDNA3, pCI, VR1012, and VR1020. This construct is used as avaccine.

Each of the newly identified genes, ProA, ProB, mmpA, the cytochromeoxidase homolog, or the partial lipoprotein signal peptidase homolog canbe used to create a DNA vaccine (reviewed in Robinson, 1997). Inaddition, any immunologically active portion of these proteins is apotential candidate for the vaccine. A plasmid containing one of thesegenes in an expression vector is constructed. The gene must be insertedin the correct orientation in order for the genes to be expressed underthe control of eukaryotic promoters. Possible promoters include, but arenot limited to, the cytomegalovirus (CMV) immediate early promoter, thehuman tissue plasminogen activator (t-PA) gene (characterized in Degenet al., 1986), and the promoter/enhancer region of the human elongationfactor alpha (EF-1 α) (characterized in Uetsuki et al., 1989).Orientation is identified by restriction endonuclease digestion and DNAsequencing.

Expression of these gene products is confirmed by indirectimmunofluorcscent staining of transiently transfected COS cells, CHOcells, or other suitable cells. The same plasmid without these genes isused as a control. Plasmid DNA is transformed into Escherichia coliDH5α. DNA is purified by cesium chloride gradients and the concentrationis determined by a standard protocol being known in the art, andincorporated by reference (Nyika et al., 1998).

Once the DNA is purified, the vector containing the insert DNA can besuspended in phosphate buffer saline solution and directly injected intodogs. Inoculation can be done via the muscle with a needle orintraveneously. Alternatively, a gene gun can be used to transportDNA-coated gold beads into cells by a technique known in the art, andhereby incorporated by reference (Fynan et al., 1993). The rationalebehind this type of vaccine is that the inoculated host expresses theplasmid DNA in its cells, and produces a protein that raises an immuneresponse. Each of the newly identified genes can be used to create avaccine by this technique.

CpG molecules can be used as an adjuvant in the vaccine. This techniqueis known in the art, and is hereby incorporated by reference (Klinman etal., 1997). Adjuvants are materials that help antigens or increase theimmune response to an antigen. The motifs consist of an unmethylated CpGdinucleotide flanked by two 5′ purines and two 3′ pyrimidines.Oligonucleotides containing CpG motifs have been shown to activate theimmune system, thereby boosting an antigen-specific immune response.This effect can be utilized in this invention by mixing the CpGoligonucleotides with the DNA vaccine, or physically linking the CpGmotifs to the plasmid DNA.

Immunofluorescence staining of E. canis in DH82 cells with rabbitanti-rProA and anti-rProB sera was performed, both ProB and ProAantiserum strongly label the intracellular ehrlichial organisms, showingthat ProA and ProB can serve as target antigens and that anti-rProA andanti-rProB sera can be used for indirect immunofluorescent assays (IFA)diagnosis, making the DNA vaccine a viable option to combat thisdisease.

Recombinant Vaccine

In order to develop a recombinant vaccine, each of the genes isindividually subcloned into overexpression vectors, and then purifiedfor vaccine development. ProA, ProB, mmpA, the cytochrome oxidasehomolog, or the partial lipoprotein signal peptidase homolog isexpressed in a plasmid with a strong promoter such as the tac, T5, or T7promoter. Alternatively, immunologically active fragments of theseproteins are used in the development of a vaccine. Each of these genesis subcloned into a plasmid and transformed into an E. coli strain asdescribed above.

The recombinant protein is overexpressed using a vector with a strongpromoter. Vectors for use in this technique include pREST (InvitrogenInc., Calif.), pKK233-3 (Pharmacia, Calif.), and the pET system(Promega, Wis.), although any vector with a strong promoter can be used.After overexpression, the proteins are purified and mixed with adjuvant.Potential adjuvants include, but are not limited to, aluminum hydroxide,QuilA, or Montamide. The purified protein is used as immunogen tovaccinate dogs by a technique being known in the art, and incorporatedby reference (Chang et al., 1993c; Chang et al., 1995). Briefly, theindividual protein is expressed and purified from E. coli. Then, thedogs are injected intramuscularly or subcutaneously with the purifiedrecombinant vaccine and adjuvant. This injection elicits an immuneresponse.

T Cell Epitope Vaccine

Direct cell cytotoxicity mediated by CD8⁺ T lymphocytes (CTL) is themajor mechanism of defense against intracellular pathogens. Theseeffector lymphocytes eliminate infected cells by recognizing shortpeptides associated with MHC class I molecules on the cell surface.Exogenous antigens enter the endosomal pathway and are presented to CD4⁺T cells in association with class II molecules whereas endogenouslysynthesized antigens are presented to CD8⁺ T cells in association withMHC class I molecules. E. canis is an intracellular pathogen thatresides in monocytes and macrophages. The present invention developsnovel ways of generating an E. canis-specific CTL response that wouldeliminate the organism from monocytes or macrophages of infectedanimals.

A strategy for increasing the protective response of a protein vaccineis to immunize with selective epitopes of the protein. The rationalebehind this is that an epitope vaccine contains the most relevantimmunogenic peptide components without the irrelevant portions.Therefore, a search is performed for the most highly antigenic portionsof the newly identified proteins.

To identify T-cell epitopes from the newly discovered proteins, aninitial electronic search for homologous sequences to known T-cellepitopes is performed. In addition, extensive T-cell epitope mapping iscarried out. Each of the proteins, ProA, ProB, mmpA, the cytochromeoxidase homolog, and the partial lipoprotein signal peptidase homolog,is tested for immunogenic peptide fragments. Mapping of T cell epitopesby a technique known in the art is hereby incorporated by reference(Launois et al., 1994; Lee and Horwitz, 1999). Briefly, short,overlapping peptide sequences (9-20 amino acids) are synthesized overthe entire length of the protein in question. These short peptidefragments are tested using healthy dogs, which have been immunized withthe protein of interest. Peripheral blood mononuclear cells from thedogs are tested for T cell stimulatory and IFN-γ inducing properties.Those fragments which elicit the strongest response are the bestcandidates for a T-cell epitope vaccine.

Once fragments are identified which will make the best epitopes, arecombinant adenylate cyclase of Bordetella bronchiseptica isconstructed carrying an E. canis CD8⁺ T cell epitope. The adenylatecyclase toxin (CyaA) of Bordetella bronchiseptica causes disease in dogsand elicits an immune response. In addition, CyaA is well suited forintracytoplasmic targeting. Its catalytic domain (AC), corresponding tothe N-terminal 400 amino acid residues of the 1,706-residue-longprotein, can be delivered to many eukaryotic cells, including cells ofthe immune system. Also, toxin internalization is independent ofreceptor-mediated endocytosis, suggesting that the catalytic domain canbe delivered directly to the cytosol of target cells through thecytoplasmic membrane. The Pseudomonas aeruginosa exotoxin A (PE) isanother toxin which could be used in this procedure to deliver peptidesor proteins into cells, by a technique known in the art, and herebyincorporated by reference (Donnelly et al., 1993).

Foreign peptides (16 residues) have been inserted into various sites ofthe AC domain of CyaA without altering its stability or catalytic andcalmodulin-binding properties. Thus, protein engineering allows thedesign and delivery of antigens that specifically stimulate CTLs. Theinduction of specific CD8⁺ T cells can play an important role in canineehrlichiosis control due to the intracellular persistence of E. canis inmonocytes.

The adenylate cyclase (AC) toxin (cya) gene of B. bronchiseptica hasbeen cloned. A synthetic double-stranded oligonucleotide encoding a 9 to20 amino acid class IT cell epitope of either ProA, ProB, mmpA, thecytochrome oxidase homolog, or the partial lipoprotein signal peptidasehomolog, is designed according to B. bronchiseptica codon usage. Thecomplementary oligonucleotides are inserted in the hypervariable regionof the cloned AC-coding sequence of the cya. This technique is known inthe art in other systems, and is incorporated by reference (Sebo et al.,1995; Guermonprez et al., 1999).

Recombinant plasmids carrying the chimeric cya gene are sequenced todetermine the copy number and orientation of the inserted epitope. Aplasmid with a complete copy of the insert that specifies the T-cellepitope (CD8⁺) in the correct orientation is chosen from the sequencedplasmids. The ability of the new chimeric protein to enter eukaryoticcells is necessary to ensure intracellular targeting of the epitopes(Fayolle et al., 1996).

A vaccine can be created in one of two ways. Recombinant chimericprotein can be purified and used to inoculate dogs. Alternatively, anattenuated B. bronchiseptica strain that carries a T-cell epitope or E.canis gene by in-frame insertion into adenylate cyclase is created byallelic-exchange. Allelic-exchange is a technique known in the art, andis hereby incorporated by reference (Cotter and Miller, 1994).

Finally, protection against E. canis infection in dogs vaccinated withthe adenylase cyclase- ProA, ProB, mmpA, cytochrome oxidase homolog, orlipoprotein signal peptidase homolog chimeric protein is determined.Wild type and recombinant ACs and CyAs are diluted to workingconcentrations in PBS and the chimeric protein is injected into dogseither intramuscularly or subcutaneously. Alternatively, the T-cellepitope is inserted into the adenylate cyclase gene of an attenuated B.bronchiseptica strain in frame; and the dogs are given the livebacteria.

Recombinant antigens are promising candidates for human and animalvaccination against various pathogens. However, a serious drawback isthe poor immunogenicity of recombinant antigens as compared to nativeantigens. A major challenge in the development of a new recombinantvaccine is, therefore, to have a new adjuvant system that increases theimmunogenicity of antigens. Cytokines are powerful immunoregulatorymolecules. Cytokines which could be used as adjuvants in this inventioninclude, but are not limited to, IL-12 (interleukin-12), GM-CSF(granulocyte-macrophage colony stimulating factor), IL-1β(interleukin-1β) and γ-IFN (gamma interferon).

These cytokines can have negative side effects including pyrogenicand/or proinflammatory symptoms in the vaccinated host. Therefore, toavoid the side effects of a whole cytokine protein, an alternateapproach is to use synthetic peptide fragments with the desiredimmunostimulatory properties. The nonapeptide sequence VQGEESNDK of IL-protein is endowed with powerful immuno-enhancing properties, and isdiscussed here to illustrate the use of a cytokine to increaseimmunogenicity.

This nonapeptide is inserted into the ProA, ProB, mmpA, the cytochromeoxidase homolog, or the partial lipoprotein signal peptidase homologprotein and its immunogenicity is compared to that of the nativeprotein. Reportedly, the insertion of this sequence into a poorlyimmunogenic recombinant antigen increases the chance of a strongprotective immune response after vaccination. This peptide could enhancethe in vivo immune response against both T-dependent and T-independentantigens. The canine IL-1β sequence may mimic many immunomodulatoryactivities of the entire molecule of IL-1β while apparently lacking manyof its undesirable proinflammatory properties. This strategy is employedto increase the immunogenicity of ProA, ProB, mmpA, cytochrome oxidase,the partial lipoprotein signal peptidase homolog and other E. canisantigens.

Plasmid pYFC199 is derived from a pBR322 plasmid by the insertion of afragment that includes the ProA, ProB, mmpA, the cytochrome oxidasehomolog, or the partial lipoprotein signal peptidase protein from E.canis. This plasmid contains a unique HindIII site where in-Frameinsertions encoding exogenous sequences can be inserted. Twocomplementary oligonucleotides, SEQ. ID. NO. 12 and SEQ. ID. NO. 13,that encode the canine IL-1β 163-171 peptide are annealed, cut withHindIII, and inserted into the pYFC199 Hind III site. The recombinantplasmid carrying the chimeric IL-1β gene is sequenced to determine theorientation of the inserted epitope.

The efficacy of the recombinant proteins as vaccines is tested in dogs.The purified protein is injected intraperitoneally into dogs. Specificpathogen free (SPF) dogs are divided into five groups: one group isgiven recombinant adenylate cyclase of Bordetella bronchisepticacarrying E. canis CD8⁺ T cell epitopes derived from ProA, ProB, mmpA,cytochrome oxidase homolog, or the partial lipoprotein signal peptidasehomolog, one group is given recombinant adenylate cyclase of Bordetellabronchiseptica as a control, one group is given the ProA, ProB, mmpA,cytochrome oxidase homolog, or the partial lipoprotein signal peptidasehomolog protein plus a canine IL-1β 163-171 insert, one group is given aT cell epitope derived from ProA, ProB, mmpA, cytochrome oxidasehomolog, or the partial lipoprotein signal peptidase homolog alone, andthe last group is given PBS as a negative control.

All animals are vaccinated (30-40 μg each) two times. The dogs arechallenged ten days after the last vaccination with 10⁷ E. canis. At dayfive postchallenge, approximately 1 ml blood from each dog is collectedin an EDTA lube. Whether the vaccinated groups eliminate the organismsas compared to that of the control group is tested by culture and PCR.Two primers derived from the genes cloned can be used to amplify thegene product from the tissues or blood samples from these dogs. Theinternal primer can also be designed for use as an oligonucleotide probeto hybridize the PCR gene product.

This invention provides a badly needed vaccine against the E. canisbacterium. The vaccine can be used to protect dogs throughout the worldfrom canine monocytic ehrlichiosis.

Accordingly, it is to be understood that the embodiments of theinvention herein described are merely illustrative of the application ofthe principles of the invention. Reference herein to details of theillustrated embodiments are not intended to limit the scope of theclaims, which themselves recite those features regarded as essential tothe invention.

1-2. (canceled)
 3. A recombinant protein comprising a protein selectedfrom the group consisting of: a) a protein having the amino acidsequence as shown in SEQ. ID. NO. 3 wherein the protein elicits animmune response against E. canis; b) a protein having the amino acidsequence as shown in SEQ. ID. NO. 5 wherein the protein elicits animmune response against E. canis: c) a protein having the amino acidsequence as shown in SEQ. ID. NO. 7 wherein the protein elicits animmune response against E. canis: d) a protein having the amino acidsequence as shown in SEQ. ID. NO. 9 wherein the protein elicits animmune response against E. canis: and e) a protein having the amino acidsequence as shown in SEQ. ID. NO. 11 wherein the protein elicits animmune response against E. canis. 4-11. (canceled)
 12. The A vaccinecomprising: a) a recombinant protein that is selected from the groupconsisting of: i) a protein having the amino acid sequence as shown inSEQ. ID. NO. 3 wherein the protein elicits an immune response against E.canis; ii) a protein having the amino acid sequence as shown in SEQ. ID.NO. 5 wherein the protein elicits an immune response against E. canis;iii) a protein having the amino acid sequence as shown in SEQ. ID. NO. 7wherein the protein elicits an immune response against E. canis; iv) aprotein having the amino acid sequence as shown in SEQ. ID. NO. 9wherein the protein elicits an immune response against E. canis; and v)a protein having the amino acid sequence as shown in SEQ. ID. NO. 11wherein the protein elicits an immune response against E. canis.
 13. Thevaccine of claim 12, wherein said vaccine further comprises an adjuvant.14-15. (canceled)
 16. The vaccine of claim 12 wherein said vaccine isadministered into a host by a method selected from the group consistingof: a) intramuscular injection; and b) subcutaneous injection.
 17. Thevaccine of claim 16 wherein said host is a dog.
 18. The vaccine of claim12 comprising a recombinant protein that includes a T cell epitopewherein said T cell epitope comprises an amino acid peptide fragment ofa protein selected from the group consisting of: a) a protein having theamino acid sequence as shown in SEQ. ID. NO. 3 wherein the proteinelicits an immune response against E. canis; b) a protein having theamino acid sequence as shown in SEQ. ID. NO. 5 wherein the proteinelicits an immune response against E. canis: c) a protein having theamino acid sequence as shown in SEQ. ID. NO. 7 wherein the proteinelicits an immune response against E. canis; d) a protein having theamino acid sequence as shown in SEQ. ID. NO. 9 wherein the proteinelicits an immune response against E. canis; and e) a protein having theamino acid sequence as shown in SEQ. ID. NO. 11 wherein the proteinelicits an immune response against E. canis.
 19. The vaccine of claim 18wherein said amino acid peptide fragment comprises nine to twenty aminoacids.
 20. The vaccine of claim 18 further comprising a recombinant DNAencoding a protein which is capable of being internalized intoeukaryotic cells.
 21. The vaccine of claim 20 wherein said proteincapable of being internalized into eukaryotic cells comprises a toxinselected from the group consisting of: a) a recombinant adenylatecyclase of Bordetella bronchiseptica; and b) a recombinant exotoxin A(PE) of Pseudomonas aeruginosa.
 22. The vaccine of claim 18 wherein saidvaccine is administered into a host by a method selected from the groupconsisting of: a) intramuscular injection; and b) subcutaneousinjection.
 23. The vaccine of claim 22 wherein said host is a dog.24-31. (canceled)
 32. A method of creating a vaccine against E. caniscomprising: a) selecting a vector capable of expressing a recombinantprotein inserted into said vector; b) insertion of a recombinant DNAinto said vector such that said recombinant protein is expressed whensaid vector is transformed into a bacterial strain wherein said DNA isselected from the group consisting of: i) a recombinant DNA that encodesa protein having m the amino acid sequence as shown in SEQ. ID. NO. 3;ii) a recombinant DNA that encodes a protein having as the amino acidsequence as shown in SEQ. ID. NO. 5; iii) a recombinant DNA that encodesa protein having as the amino acid sequence as shown in SEQ. ID. NO. 7;iv) a recombinant DNA that encodes a protein having the amino acidsequence as shown in SEQ. ID. NO. 9; and v) a recombinant DNA thatencodes a protein having the amino acid sequence as shown in SEQ. ID.NO. 11; and c) harvesting said recombinant protein from said bacterialstrain.
 33. The method of claim 32, wherein said vaccine furthercomprises an adjuvant.
 34. The method of claim 32, wherein said vaccinefurther comprises a promoter selected from the group consisting of:a)tac; b) T5; and c)T7.
 35. The method of claim 32, wherein saidbacterial strain is E. coli.
 36. The method of claim 32, wherein saidvector is selected from the group consisting of: a) pREST; b) pET; andc) pKK233-3. 37-38. (canceled)
 39. The method of claim 32 wherein saidvaccine is injected into said host in a manner selected from the groupconsisting of: a) intramuscular injection; and b) subcutaneousinjection.
 40. The method of claim 39 wherein said host is a dog. 41-65.(canceled)
 66. A vaccine comprising a DNA selected from the groupconsisting of a) a recombinant DNA that encodes a protein having theamino acid sequence as shown in SEQ. ID. NO. 3 wherein the proteinelicits an immune response against E. canis; b) a recombinant DNA thatencodes a protein having the amino acid sequence as shown in SEQ. ID.NO. 5 wherein the protein elicits an immune response against E. canis;c) a recombinant DNA that encodes a protein having the amino acidsequence as shown in SEQ. ID. NO. 7 wherein the protein elicits animmune response against E. canis; d) a recombinant DNA that encodes aprotein having the amino acid sequence as shown in SEQ. ID. NO. 9wherein the protein elicits an immune response against E. canis; and e)a recombinant DNA that encodes a protein having the amino acid sequenceas shown in SEQ. ID. NO. 11 wherein the protein elicits an immuneresponse against E. canis.
 67. The vaccine of claim 66, wherein the DNAis selected from the group consisting of: a) SEQ. ID. NO. 2, whereinSEQ. ID. NO. 2encodes a protein having the amino acid sequence as shownin SEQ. ID. NO. 3 and wherein the protein elicits an immune responseagainst E. canis; b) SEQ. ID. NO. 4, wherein SEQ. ID. NO. 4 encodes aprotein having the amino acid sequence as shown in SEQ. ID. NO. 5 andwherein the protein elicits an immune response against E. canis; c) SEQ.ID. NO. 6, wherein SEQ. ID. NO. 6 encodes a protein having the aminoacid sequence as shown in SEQ. ID. NO. 7 and wherein the protein elicitsan immune response against E. canis; d) SEQ. ID. NO. 8, wherein SEQ. ID.NO. 8 encodes a protein having the amino acid sequence as shown in SEQ.ID. NO. 9 and wherein the protein elicits an immune response against E.canis; and e) SEQ. ID. NO. 10, wherein SEQ. ID. NO. 10 encodes a proteinhaving the amino acid sequence as shown in SEQ. ID. NO. 11 and whereinthe protein elicits an immune response against E. canis.
 68. A vaccinecomprising a vector, the vector capable of expressing an isolatedrecombinant DNA comprising the isolated recombinant DNA inserted intothe vector such that a recombinant protein is expressed when the vectoris provided in an appropriate host wherein the isolated recombinant DNAis selected from the group consisting of: a) SEQ. ID. NO. 2, whereinSEQ. ID. NO. 2encodes a protein having the amino acid sequence as shownin SEQ. ID. NO. 3 and wherein the protein elicits an immune responseagainst E. canis; b) SEQ. ID. NO. 4, wherein SEQ. ID. NO. 4 encodes aprotein having the amino acid sequence as shown in SEQ. ID. NO. 5 andwherein the protein elicits an immune response against E. canis; c) SEQ.ID. NO. 6, wherein SEQ. ID. NO. 6 encodes a protein having the aminoacid sequence as shown in SEQ. ID. NO. 7 and wherein the protein elicitsan immune response against E. canis; d) SEQ. ID. NO. 8, wherein SEQ. ID.NO. 8 encodes a protein having the amino acid sequence as shown in SEQ.ID. NO. 9 and wherein the protein elicits an immune response against E,canis; and e) SEQ. ID. NO. 10, wherein SEQ. ID. NO. 10 encodes a proteinhaving the amino acid sequence as shown in SEQ. ID. NO. 11 and whereinthe protein elicits an immune response against E. canis.